Proteolysis-targeting chimera (PROTAC) molecules have emerged as a revolutionary approach in drug discovery, offering the potential to selectively degrade target proteins. However, their design, characterized by a bivalent structure comprising a target protein-binding ligand (warhead), an E3 ligase-binding ligand, and a linker, often results in physicochemical properties that deviate significantly from traditional small-molecule drugs. These deviations present substantial challenges in drug development, particularly in achieving oral bioavailability, which is a critical attribute for many therapeutic applications. This article is a practical perspective on the ADME challenges encountered during PROTAC development.
1 PROTACs Unique Property Challenges
1.1 Plasma Stability
PROTAC molecules, particularly those containing labile amide or ester bonds within the linker or warhead regions, can exhibit poor plasma stability. Metabolic identification (metID) studies are crucial to pinpoint metabolic hotspots and guide structural modifications to enhance stability.
1.2 Water Solubility
Poor water solubility is a common issue with PROTACs, complicating formulation development for in vivo studies. A range of solvents, including DMSO, PEG400, and Solutol-15, are often employed, each with its own limitations (e.g., Solutol-15 toxicity in rats). pH adjustment may be necessary for PROTACs with ionizable groups.
1.3 Lipophilicity (logP)
The bivalent nature of PROTACs leads to inherently high logP values. While high lipophilicity can enhance membrane permeability, it also increases the risk of non-specific binding, potentially affecting the reliability of in vitro assays such as liver microsomal stability.
1.4 Chirality
PROTAC molecules frequently possess multiple chiral centers, resulting in a complex mixture of stereoisomers. Chiral separation and in vivo interconversion studies are essential to determine the optimal stereochemical configuration and avoid costly development of multiple isomers.
2 In vitro ADME studies: Protein Binding and DDI
2.1 Plasma Protein Binding (PPB)
PROTACs typically exhibit high PPB, often exceeding 90%, with molecules above 900 Da occasionally reaching PPB > 99%. For compounds with PPB > 99%, the FDA recommends using 99% for in vivo efficacy predictions. Equilibrium dialysis and supercentrifugation are common methods. While supercentrifugation minimizes membrane adsorption, it does not allow for recovery rate calculation. Pre-incubation of the dialysis membranes can mitigate adsorption issues in equilibrium dialysis. The choice of method depends on the specific properties of the PROTAC molecule.
2.2 Media Protein Binding
Protein binding in cell culture media (e.g., 10% FBS) is typically in the range of 70-90%. Accurate determination of unbound drug concentration in the media is crucial for predicting in vivo effective/efficacious doses. Using uncorrected in vitro IC50 values can lead to significant overestimation of the required in vivo dose.
2.3 The Caco-2 Model
The Caco-2 cell permeability assay, a widely used tool for predicting oral absorption of small molecules, often fails to accurately predict the in vivo oral bioavailability of PROTACs. PROTACs, being beyond the rule of five (bRo5) compounds, exhibit slow intracellular permeation, leading to underestimation of permeability coefficients (Papp) despite acceptable in vivo absorption. High non-specific binding further complicates Caco-2 permeability measurements. Alternative in vitro permeability assays tailored for PROTACs are needed.
2.4 Liver Microsomal Stability
Due to PROTACs’ high adsorption potential, microsomal protein concentrations should be kept below 0.5 mg/mL to avoid underestimating clearance. While microsomal protein binding is not routinely measured, it becomes necessary when using in vitro-in vivo extrapolation (IVIVE) to predict in vivo clearance. Assessing microsomal metabolism (rate and NADPH dependence) is essential.
2.5 Penetration vs. Permeability
Cellular or tissue penetration, referring to the ability of a drug to cross a single cell membrane, should not be confused with permeability, which describes the ability to cross a cellular barrier (e.g., intestinal epithelium, blood-brain barrier). PROTACs generally exhibit good cellular uptake and tissue distribution (high volume of distribution, Vd). In contrast, permeability, as measured by Papp, is often low for PROTACs, which explains the disconnect between low Caco-2 permeability and acceptable in vivo oral absorption.
2.6 Drug-Drug Interaction (DDI) Potential
The warhead component of a PROTAC may exhibit CYP inhibition, which can be exacerbated in the PROTAC molecule, increasing the risk of DDI. Conversely, minimizing the DDI potential of the warhead and E3 ligase binding ligand can mitigate the overall DDI risk of the PROTAC. CYP time-dependent inhibition (TDI) is a critical consideration.
Conclusion
PROTAC molecules present a unique set of ADME challenges that must be carefully addressed to achieve successful oral drug development. These challenges include plasma instability, poor water solubility, high lipophilicity, complex stereochemistry, DDI potential, and poor oral bioavailability. Traditional in vitro models like Caco-2 have limitations in predicting the in vivo behavior of PROTACs. A thorough understanding of protein binding, including plasma and media binding, and the distinction between cellular penetration and permeability are crucial. By addressing these challenges, researchers can pave the way for the development of orally bioavailable PROTAC therapeutics.